Alexa Fluor 647 AffiniPure Donkey Anti-Mouse IgG (H+L)

What is Alexa Fluor 647 AffiniPure Donkey Anti-Mouse IgG (H+L) and how does it function in immunoassays?

Alexa Fluor 647 AffiniPure Donkey Anti-Mouse IgG (H+L) is a secondary antibody generated using mouse IgG as the immunogen and subsequently conjugated to the Alexa Fluor 647 fluorescent dye . It functions by specifically binding to mouse IgG primary antibodies in immunoassay applications, allowing for the detection of target antigens. The antibody recognizes both the heavy and light chains (H+L) of mouse IgG, as well as the light chains of other mouse immunoglobulins . Based on immunoelectrophoresis and ELISA testing, this antibody reacts with the whole molecule of mouse IgG while showing minimal cross-reactivity with non-immunoglobulin serum proteins or immunoglobulins from other specified species .

What are the spectral properties of Alexa Fluor 647 and how do they influence experimental design?

Alexa Fluor 647 has distinct spectral characteristics that researchers must consider when designing experiments:

The far-red emission profile of Alexa Fluor 647 offers significant advantages in biological research, particularly the low autofluorescence of biological specimens in this spectral region . This property enhances signal-to-noise ratios and improves detection sensitivity for cellular and subcellular targets. When designing multicolor experiments, these spectral properties enable effective separation from fluorophores in the green and yellow-orange ranges .

What research applications is Alexa Fluor 647 AffiniPure Donkey Anti-Mouse IgG (H+L) suited for?

This antibody is validated for numerous research applications across cellular and molecular biology:

• Flow Cytometry: Provides excellent signal separation and brightness for quantitative analysis of cellular populations .

• Immunofluorescence: Enables high-resolution imaging of cellular and subcellular targets in fixed samples .

• Immunocytochemistry: Facilitates detection of antigens in cultured cells with superior signal-to-background ratio .

• Immunohistochemistry: Supports both frozen and paraffin-embedded section analysis with minimal autofluorescence interference .

• ELISA: Allows sensitive detection of antigen-antibody interactions in solution-based assays .

• Confocal Microscopy: Particularly well-suited for multicolor detection due to its spectral properties and brightness .

Methodologically, the antibody performs optimally when used at dilutions of 1:100-1:800 for flow cytometry and histochemistry/cytochemistry applications, though optimal concentrations should be determined empirically for each application and experimental system .

How does the specificity of this antibody affect experimental design, and what cross-reactivity considerations must be addressed?

The "MinX" designation in Alexa Fluor 647 AffiniPure Donkey Anti-Mouse IgG (H+L) indicates it has been adsorbed against multiple species to minimize cross-reactivity . Specifically, this antibody shows minimal cross-reactivity with bovine, chicken, goat, guinea pig, Syrian hamster, horse, human, rabbit, and sheep proteins . This high specificity reduces background signal in experiments where these species' proteins may be present.

What methodological considerations affect the optimal dilution and incubation conditions for this antibody?

Establishing optimal working conditions for Alexa Fluor 647 AffiniPure Donkey Anti-Mouse IgG (H+L) requires systematic titration and validation. While the manufacturer recommends dilutions of 1:100-1:800 for flow cytometry and histochemistry applications , the optimal concentration depends on:

• Target abundance: Lower abundance targets may require higher antibody concentrations

• Sample type: Different tissue types and fixation methods affect antibody penetration and binding

• Incubation time and temperature: Longer incubations at lower temperatures often improve signal-to-noise ratios

• Buffer composition: Addition of blocking proteins and detergents can reduce background staining

A methodological approach to optimization involves:

The optimal protocol should yield maximum specific signal with minimal background staining, and these conditions should be validated for each experimental system and application .

How should Alexa Fluor 647 AffiniPure Donkey Anti-Mouse IgG (H+L) be stored and handled to maintain optimal performance?

Proper storage and handling of this antibody are critical for maintaining its immunoreactivity and fluorescence properties over time. The antibody is typically supplied as a lyophilized powder in a buffer containing 0.05% NaN3, 15 mg/ml BSA, and 250 mM NaCl in 10 mM PBS (pH 7.6) .

Storage recommendations:

• Store lyophilized antibody at 2-8°C in the dark

• After reconstitution, store in small aliquots at -20°C to avoid repeated freeze-thaw cycles

• Protect from prolonged exposure to light to prevent photobleaching of the Alexa Fluor 647 dye

• Use within the specified expiration date

Working with the antibody:

• Allow solutions to equilibrate to room temperature before opening

• Centrifuge briefly before opening to ensure all material is at the bottom of the tube

• Prepare working dilutions on the day of use for optimal results

• Minimize exposure to light during handling and incubation steps

Following these methodological guidelines helps preserve antibody functionality and fluorescence intensity, ensuring consistent experimental results over time.

How does Alexa Fluor 647 compare to other far-red fluorophores for multicolor imaging and flow cytometry?

Alexa Fluor 647 offers several advantages over alternative far-red fluorophores, which influences its selection for complex multicolor experiments:

Methodologically, Alexa Fluor 647 is particularly advantageous for:

• Confocal microscopy applications requiring extended imaging periods, due to its superior photostability

• Multicolor flow cytometry panels, where its brightness facilitates detection of low-abundance antigens

• Studies involving tissue sections with high autofluorescence, as the far-red emission minimizes interference from endogenous fluorophores

• Applications requiring cost-effective alternatives to allophycocyanin (APC) conjugates while maintaining similar detection sensitivity

When designing complex multicolor panels, Alexa Fluor 647's spectral profile allows effective combination with fluorophores in the blue, green, and yellow-orange ranges without significant spectral overlap concerns .

What are the optimal imaging systems and configuration parameters for detecting Alexa Fluor 647 signals?

Detecting Alexa Fluor 647 requires specific instrumentation and configuration parameters for optimal results:

Confocal Microscopy Setup:

• Excitation source: 633-647 nm laser line (optimal)

• Detection range: 665-680 nm bandpass filter

• Pinhole setting: 1-1.2 Airy units for optimal resolution/signal balance

• Detector gain: Calibrated to maximize signal while avoiding saturation

Flow Cytometry Configuration:

• Excitation laser: 633-640 nm

• Detection filter: 660/20 bandpass

• Compensation: Minimal when paired with fluorophores below 600 nm

Important methodological considerations:

• Alexa Fluor 647 is not optimally visualized using conventional epifluorescence microscopes with mercury lamps due to insufficient excitation at required wavelengths

• The dye cannot be well visualized by eye due to its far-red emission (667 nm), necessitating digital imaging systems

• For optimal detection, confocal microscopes equipped with appropriate lasers and far-red detectors are recommended

• In flow cytometry, Alexa Fluor 647 and APC-conjugated antibodies are interchangeable in most configurations, providing flexibility in panel design

These instrument-specific parameters ensure maximum signal detection sensitivity while minimizing background and cross-talk with other fluorophores in multicolor experiments.

How can Alexa Fluor 647 AffiniPure Donkey Anti-Mouse IgG (H+L) be integrated into complex multi-labeling experiments?

Designing complex multi-labeling experiments with Alexa Fluor 647 AffiniPure Donkey Anti-Mouse IgG (H+L) requires careful consideration of primary antibody combinations, fluorophore selection, and experimental workflow:

Strategic Experimental Design Approach:

• Primary Antibody Selection:

  • Choose mouse-origin primary antibodies for targets to be visualized with Alexa Fluor 647

  • Select primary antibodies from different host species (rabbit, goat, etc.) for other targets

  • Consider using isotype-specific secondary antibodies when multiple mouse primaries are needed

• Complementary Fluorophore Selection:

  • Pair Alexa Fluor 647 with fluorophores having minimal spectral overlap:

    • Alexa Fluor 488 (Ex/Em: 496/519 nm)

    • Alexa Fluor 555 (Ex/Em: 555/565 nm)

    • Alexa Fluor 594 (Ex/Em: 590/617 nm)

• Sequential Staining Protocol:
  For highly sensitive multi-labeling experiments, a sequential approach may be optimal:

  • Apply first primary antibody, wash thoroughly

  • Apply corresponding secondary antibody (e.g., Alexa Fluor 647 anti-mouse), wash thoroughly

  • Block remaining mouse epitopes with excess unconjugated anti-mouse IgG

  • Proceed with additional primary-secondary antibody pairs

This methodological approach minimizes cross-reactivity and ensures accurate multi-target visualization . When properly implemented, this strategy allows for precise localization of multiple cellular components with minimal false co-localization artifacts.

What are common issues with Alexa Fluor 647 detection and their methodological solutions?

Researchers may encounter several challenges when working with Alexa Fluor 647 AffiniPure Donkey Anti-Mouse IgG (H+L). Here are systematic approaches to identifying and resolving these issues:

When troubleshooting experiments with this antibody, a systematic approach involves:

• Testing positive and negative controls to verify antibody functionality

• Performing serial dilutions to determine optimal concentration

• Comparing different blocking reagents to minimize background

• Evaluating alternative fixation methods if antigen accessibility is compromised

These methodological adjustments should be documented and evaluated quantitatively to establish reproducible protocols for specific experimental systems .

How can researchers validate the specificity of staining patterns observed with this antibody?

Validating the specificity of Alexa Fluor 647 AffiniPure Donkey Anti-Mouse IgG (H+L) staining is critical for generating reliable research data. A comprehensive validation approach includes:

Essential Validation Controls:

• Negative Controls:

  • Secondary antibody-only control (omit primary antibody)

  • Isotype control (irrelevant mouse IgG primary + Alexa Fluor 647 secondary)

  • Blocking peptide competition (pre-incubate primary with excess target peptide)

• Positive Controls:

  • Known positive samples with established expression patterns

  • Comparison with alternative detection methods (Western blot, qPCR)

  • Parallel staining with alternative antibody against same target

• Specificity Tests:

  • Signal ablation following target knockdown (siRNA/shRNA)

  • Absence of signal in knockout models

  • Co-localization with alternative markers of the same structure/protein

• Cross-Reactivity Assessment:

  • Test staining in tissues/cells known to lack the target

  • Evaluate signal in samples from different species

  • Compare staining pattern with published literature

Methodologically, researchers should document all validation steps, quantify signal-to-background ratios, and establish clear criteria for distinguishing specific from non-specific staining. This rigorous approach ensures that experimental observations reflect true biological phenomena rather than technical artifacts .

What strategies can improve signal-to-noise ratio when working with challenging samples?

Maximizing signal-to-noise ratio with Alexa Fluor 647 AffiniPure Donkey Anti-Mouse IgG (H+L) in challenging samples requires advanced methodological approaches:

Signal Enhancement Strategies:

• Antigen Retrieval Optimization:

  • Test multiple retrieval methods (heat-induced vs. enzymatic)

  • Optimize retrieval buffer composition (citrate pH 6.0 vs. EDTA pH 9.0)

  • Determine optimal retrieval duration for specific tissues

• Signal Amplification Techniques:

  • Tyramide signal amplification (10-50× signal enhancement)

  • Multilayer detection systems (biotin-streptavidin)

  • Sequential application of multiple secondary antibodies

• Background Reduction Methods:

  • Extended blocking (overnight at 4°C with 5-10% serum/BSA)

  • Use of specialized blocking reagents (Mouse-on-Mouse blocking for mouse tissues)

  • Pre-adsorption of secondary antibody with tissue powder from relevant species

• Autofluorescence Management:

  • Sudan Black B treatment (0.1-0.3% in 70% ethanol)

  • Copper sulfate incubation (1-10mM CuSO₄ in 50mM ammonium acetate)

  • Photobleaching pre-treatment (exposure to strong light before antibody application)

• Protocol Modifications for Specific Sample Types:

These methodological refinements should be systematically tested and optimized for each specific sample type and target, with quantitative assessment of signal-to-noise ratios to guide protocol development .

How does Alexa Fluor 647 AffiniPure Donkey Anti-Mouse IgG (H+L) compare in emerging super-resolution microscopy techniques?

Alexa Fluor 647 has become particularly valuable in super-resolution microscopy applications due to its photophysical properties. In techniques like Stochastic Optical Reconstruction Microscopy (STORM) and direct Stochastic Optical Reconstruction Microscopy (dSTORM), Alexa Fluor 647 exhibits favorable photoswitching behavior in appropriate buffer systems, enabling localization precisions of approximately 10-20 nm . The photostability of Alexa Fluor 647 also makes it suitable for Stimulated Emission Depletion (STED) microscopy, where its resistance to photobleaching allows for repeated depletion cycles.

Methodologically, researchers implementing super-resolution techniques with this antibody should:

• Use higher concentrations of primary and secondary antibodies to ensure adequate labeling density

• Prepare specialized imaging buffers containing oxygen scavenging systems and thiol compounds for STORM/dSTORM

• Optimize fixation to minimize structural alterations at the nanoscale level

• Consider using smaller probes (such as nanobodies) conjugated to Alexa Fluor 647 for improved spatial resolution

These adaptations help maximize the resolution-enhancing capabilities of Alexa Fluor 647 in advanced microscopy applications, enabling visualization of subcellular structures below the diffraction limit .

What recent research advances have expanded the utility of Alexa Fluor 647-conjugated secondary antibodies?

Recent methodological advances have expanded the applications of Alexa Fluor 647-conjugated secondary antibodies in several research domains:

• Multiplexed Imaging Techniques:
  New cyclical immunofluorescence methods allow for sequential staining, imaging, and antibody elution/removal, enabling visualization of >40 targets in the same sample using a limited set of fluorophores including Alexa Fluor 647.

• Correlative Light and Electron Microscopy (CLEM):
  Protocols combining Alexa Fluor 647 immunofluorescence with electron microscopy enable researchers to correlate fluorescence patterns with ultrastructural features at nanometer resolution.

• Intravital Imaging Applications:
  Optimized Alexa Fluor 647 conjugates with enhanced tissue penetration properties have enabled deep tissue imaging in live animal models with reduced phototoxicity.

• Quantitative Approaches:
  Development of calibration standards and analysis algorithms has improved quantitative assessment of protein expression levels using Alexa Fluor 647 fluorescence intensity.

These advances represent the cutting edge of immunofluorescence methodology and illustrate how Alexa Fluor 647 AffiniPure Donkey Anti-Mouse IgG (H+L) continues to serve as a valuable tool in advancing biological research .

What methodological considerations apply when transitioning from single-cell to tissue-level imaging with this antibody?

Transitioning from single-cell to tissue-level imaging with Alexa Fluor 647 AffiniPure Donkey Anti-Mouse IgG (H+L) requires several methodological adaptations:

Protocol Modifications for Tissue Imaging:

• Sample Preparation Adjustments:

  • Optimize fixation duration based on tissue thickness (longer for thicker sections)

  • Implement extended permeabilization steps (24-48 hours for dense tissues)

  • Consider vibratome sectioning for improved antibody penetration in thick specimens

• Antibody Concentration and Incubation:

  • Increase antibody concentration by 2-3 fold compared to cell culture applications

  • Extend incubation times (24-72 hours at 4°C for thick sections)

  • Consider using specialized tissue penetration enhancers

• Imaging Parameter Adaptations:

  • Implement optical clearing techniques compatible with Alexa Fluor 647 (CUBIC, CLARITY)

  • Utilize confocal z-stacking with appropriate step sizes for 3D reconstruction

  • Adjust laser power to balance tissue penetration with photobleaching concerns

• Signal Validation Approaches:

  • Compare staining patterns between thick sections and thin serial sections

  • Validate with alternative visualization methods (e.g., chromogenic IHC)

  • Implement computational approaches to correct for depth-dependent signal attenuation